The present invention relates to methods for expanding, activating, committing or mobilizing pluripotent self-renewing and committed stem cells.
Functionally, hematopoietic growth factors can be considered to belong to one of three groups. The first or multilineage group includes interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) which act on early colony forming units (CFU""s) including colony forming unit-granulocyte, erythrocyte, megakaryocyte, macrophage (CFU-GEMM), colony forming unit-granulocyte-macrophage (CFU-GM), burst forming units erythrocyte (BFU-E) or megakaryocytes, (BFU-MK). The second or unilineage group includes erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), interleukin 5 (IL-5), macrophage colony stimulating factor (M-CSF) and thrombopoietin (TPO), and act on later hematopoietic progenitors (i.e., colony forming unit erythrocyte (CFU-E), colony forming unit megakaryocyte (CFU-Mk), and colony forming unit eosinophil (CFU-Eo). The third or xe2x80x9cpotentiatingxe2x80x9d group includes interleukin 6 (IL-6), interleukin 11 (IL-11), lymphocyte inhibitory factor (LIF), fibroblast growth factor basic (FGFb), stem cell factor (SCF) and Flt3 ligand (Flt3-L), and act to potentiate the activities of other hematopoietic factors. Within the third group, SCF and Flt3-L both show marked activity on hematopoietic stem cells and thus have been considered special circumstance/stem cell growth factors.
G-CSF and GM-CSF are two commonly used hematopoietic growth factors. The principal action of G-CSF is the stimulation of colony forming unit granulocyte (CFU-G), which in vivo manifests into an augmented production of polymorphonuclear leukocyte (neutrophil) as well as enhancing the phagocytic and cytotoxic functions of neutrophils in general. G-CSF has been shown to be effective in the treatment of severe neutropenia following autologous bone marrow transplantation and high-dose chemotherapy. GM-CSF and G-CSF are each used to decrease the period of neutropenia seen during this type of therapy and thereby reduces morbidity secondary to bacterial and fungal infections. When used as a part of an intensive chemotherapy regimen, G-CSF can decrease the frequency of both hospitalization for febrile neutropenia and interruptions in life-saving chemotherapy protocols. G-CSF also has proven to be effective in the treatment of severe congenital neutropenias. In patients with cyclic neutropenia, G-CSF therapy, while not eliminating the neutropenic cycle, will increase the level of neutrophils and shorten the length of the cycle sufficiently to prevent recurrent infections. G-CSF therapy can improve neutrophil counts in some patients with myelodysplasia or marrow damage. The neutropenia of AIDS patients receiving AZT also can be partially or completely reversed.
G-CSF is typically administered by subcutaneous injection or intravenous infusion at a dose of 1 to 20 xcexcg/kg per day. The distribution and clearance rate from plasma (half-life of 3.5 hours) are similar for both routes of administration. A continuous, 24-hour intravenous infusion can be used to produce a steady-state serum concentration of the growth factor. As with GM-CSF therapy, G-CSF is given daily following bone marrow transplantation or intensive chemotherapy will increase granulocyte production and shorten the period of severe neutropenia. In bone marrow transplantation and intensive chemotherapy patients, continuous daily administration for 14 to 21 days or longer may be necessary to correct the neutropenia. With less intensive chemotherapy, fewer than 7 days of treatment may be needed.
Both G-CSF and GM-CSF will increase the number of marrow progenitor cells in the circulation, a particularly valuable function in patients preparing for stem cell collection. Post-transplant infusions of harvested stem cells together with G-CSF or GM-CSF may reduce the severity of the post-transplant neutropenia.
One hematopaetic growth factor that has recently received considerable attention for its unique properties is Flt3-L. Flt3-L is a transmembrane glycoprotein of approximately 30 kDa. Mouse and human Flt3-L share significant homology at the amino acid level (xcx9c70%), and show cross-species reactivity, so testing human Flt3-L in mouse produces the same or similar biological effects as would occur in the human. Cells known to express Flt3-L include human and mouse T cell lines, as well as architectural cells of the bone marrow, specifically the bone marrow fibroblast.
Some of the myelopoietic, or white blood cell potentiating effects attributed to Flt3-L include: I) an expansion of CD34+ CD38xe2x88x92 cell number when used in conjunction with SCF and IL-3; 2) an increase in high proliferative potential colony forming cells (HPP-CFC) and CFU-GM numbers; and 3) in the presence of GM-CSF, the formation of large numbers of CFU-GM. Individual and direct myelopoietic effects of Flt3-L include an increase in CFU-GM, CFU-GEMM and HPP-CFC survival and a preferential induction of macrophages under certain conditions. Flt3-L alone apparently has minimal or no effects on erythroid and megakaryocyte progenitors.
There is substantial data showing that the system of Flt3-L and its receptor also plays an important role in lymphopoiesis, the processes involved in normal growth and maturation of lymphocytes. This important activity has been confirmed in mice made deficient for Flt3-L System. In these mice hematopoietic populations are essentially normal but marked deficiencies of early B cell progenitors are found in the bone marrow. This has led to the suggestion that Flt3-L, perhaps expressed constitutively by bone marrow fibroblasts, is a normal regulator of B cell lymphopoiesis, while cytokines produced by activated lymphocytes synergize with Flt3-L in times of stress to accelerate B cell development.
In addition to its effects on hematopoietic cells and B cells, Flt3-L has also been shown to stimulate the production of dendritic cells, a highly specialized cell involved in antigen presentation and therefore, normal immunity. Also, with the observation that Flt3-L stimulates the production of dendritic cells, Flt3-L has been identified for potential use in the area of vaccines, both traditional delivery of heat killed or otherwise attenuated agents, as well as protein, peptide or DNA vaccines.
For additional information on Flt3-L, see, for example, Shurin et al., xe2x80x9cFLT3: Receptor and Ligand. Biology and Potential Clinical Applicationxe2x80x9d, Cytokine and Growth Factor Reviews, Vol. 9, No. 1, pp. 37-48, 1998.
One of the problems associated with the hematopoietic growth factors such as G-CSF, GM-CSF, SCF and Flt3-L, is the need for multiple daily injections. This, in turn leads to another common disadvantage of current injectable therapies such as these, that being the creation of a saw-toothlike effect of plasma drug levels. This is due to the creation of large bolus bursts of drug shortly after injection, leading to supraphysiologic levels of drug, followed by rapid drops in plasma drug levels as the drug is cleared from the body by normal clearance processes. Upon the next injection, the pattern is repeated with large spikes in plasma levels followed by sub-therapeutic levels until the next injection. An additional problem with current hematopoietic growth factor therapy includes fever and mild-to-moderate bone pain in patients receiving high doses over a long period. In addition, local skin reactions and mild to moderate splenomegaly have been reported.
There is a significant need for improved formulations and methods for delivery of hematopoietic growth factors that address one or more of these problems, especially as treatments involving the use of hematopoietic growth factors continue to expand.
The present invention provides a method for delivering a hematopoietic growth factor or other active agent to a host for expanding, activating, committing or mobilizing pluripotent self-renewing and committed stem cells in the host. As will be appreciated, hematopoietic growth factors are such active agents that expand, activate, commit and/or mobilize at least hematopoietic stem cells. The method of the invention is described herein with primary reference to hematopoietic growth factors, but the principles discussed apply equally to other active agents or to active agents administered for the purpose of expanding, activating, committing and/or mobilizing pluripotent self-renewing and committed stem cells other than hematopoietic stem cells.
The method of the present invention can be used to effect sustained delivery of a hematopoietic growth factor, thereby advantageously increasing the plasma half-life of of hematopoietic growth factor, and thereby also reducing the number of administrations, and therefore the number of injections, required for treatment. Moreover, the saw-tooth profiles of drug plasma levels experienced conventionally should be reduced with less frequent administrations, as should side effects caused by the frequent injections with conventional treatments. Furthermore, it has been found in at least some cases, that the activity of the hematopoietic growth factor is significantly improved with the method of the present invention, relative to conventional techniques. Therefore, not only should fewer administrations be required for a treatment program, but less hematopoietic growth factor should also be required in many instances, which would be expected to generally reduce the severity of side effects.
In one aspect, the method of the present invention for deliverying a hematopoietic growth factor involves administering to a host a hematopoietic delivery composition. matopoietic growth factor delivery composition comprising a hematopoietic growth factor, a first biocompatible polymer, a second biocompatible polymer and a liquid vehicle. The first biocompatible polymer and the liquid vehicle interact in such a manner and are present in such proportions that the composition exhibits reverse-thermal viscosity behavior, in that the viscosity of the composition increases with increasing temperature over at least some temperature range. The second biocompatible polymer is a protective colloid.
The reverse-thermal viscosity behavior of the delivery composition permits the delivery composition to be administered to a host as a lower-viscosity flowable medium, which then converts to a higher-viscosity form in vivo. The hematopoietic growth factor is then advantageously released in a sustained manner from the protective environment of the higher-viscosity form of the delivery composition. To accomplish this result, the delivery composition should exhibit reverse-thermal viscosity behavior over at least some temperature range below the physiologic temperature of the host. The presence of the second biocompatible polymer helps to protect the composition from premature degradation in vivo due to invasion by aqueous biological fluids, such as are encountered by the delivery composition inside the host after administration. The inclusion of the second biocompatible polymer, therefore, is important to help protect the delivery composition so that the delivery composition can successfully make the transition from the lower-viscosity flowable medium to the higher-viscosity form following administration. Also, the second biocompatible polymer helps to inhibit premature deterioration in vivo of the higher-viscosity form, thereby promoting a prolonged release of the hematopoietic growth factor. Surprisingly, the inclusion of the second biocompatible polymer has also resulted in an observed significant increase in the activity of the hematopoietic growth factor under at least some circumstances. Although the mechanism of this enhancement is not well understood, the enhancement in activity of the observed hematopoietic growth factor with the composition is significant and surprising.
In one embodiment, the delivery composition that is administered to the host exhibits a reverse-thermal gelation property, which is a special case of reverse-thermal viscosity behavior in which the higher-viscosity form of the delivery composition is a gel (i.e., gelatinous substance). In this preferred embodiment of the delivery composition, the composition should have a reverse-thermal liquid-gel transition temperature that is no higher than the physiologic temperature of the host. In this situation, the composition is administered to the host as a flowable medium at a chilled temperature, and as the delivery composition warms in the host following administration the delivery composition converts to the gel form. Because the gel form is typically substantially immobile, the hematopoietic growth factor is released within the host at the desired location from the protective environment of the gel to facilitate sustained delivery of the hematopoietic growth factor.
In one preferred embodiment of the method, the delivery composition is in the form of a flowable medium at least at a first temperature and in the gel form at least at a second temperature that is higher than the first temperature, but not higher than the physiologic temperature of the host. In this way, the delivery composition is delivered to the host at the first temperature when the delivery composition is in the form of a flowable medium, and after administration, the delivery composition converts to the gel form as the temperature of the delivery composition increases within the host. For example, when the delivery composition is intended for use by a human host, the first temperature could advantageously be below 20xc2x0 C., preferably in a range of 10xc2x0 C. to 20xc2x0 C., and the second temperature could advantageously be in a range of 25xc2x0 C. to 37xc2x0 C. In any event, with a human host the delivery composition should be preferably in the gel form at 37xc2x0 C. Also, at the first temperature, the first biocompatible polymer is preferably substantially entirely dissolved in the liquid vehicle in the form of a solution that is liquid and flowable to an extent to impart sufficient fluidity to the delivery composition so that the delivery composition is administrable to a host by injection. The hematopoietic growth factor may also be dissolved in the solvent, or may be in the form of a fine precipitate suspended by the hematopoietic growth factor/solvent solution. The second biocompatible polymer will typically be in the form of a xe2x80x9ccolloidal solutionxe2x80x9d in the liquid vehicle, at least at the first temperature.
Also, for enhanced performance, the hematopoietic growth factor should be uniformly dispersed throughout the gel at the time of administration, which can typically be accomplished by mixing the composition at a temperature at which the first biocompatible polymer/liquid vehicle combination is in the form of a flowable liquid solution of the first biocompatible polymer in the liquid vehicle. In this way the hematopoietic growth factor can be dissolved in or uniformly dispersed throughout the solution, and then the temperature of the composition can be raised to convert the composition to the gel form for storage prior to use.
Both the foregoing summary description and the following detailed description are exemplary and are intended to provide explanation of the invention as claimed. Other aspects and novel features will be readily apparent to those skilled in the art from the following detailed description of the invention.